Glycoprotein gIII of pseudorabies virus is multifunctional.

One of the major glycoproteins of pseudorabies virus, gIII, is nonessential for growth in cell culture. Mutants defective in gIII, however, consistently yield lower titers of infectious virus (3- to 20-fold) than does wild-type virus. The interactions of gIII- mutants with their host cells were compared with those of wild-type virus in an attempt to uncover the functions of gIII. We show that gIII plays a major role in the stable adsorption of the virus to its host cell; in the absence of gIII, the rate of adsorption is reduced and adsorption is easily reversed by washing. Thus, adsorption of pseudorabies virus can be said to occur in at least the following two ways: (i) a gIII-mediated rapid adsorption or (ii) a slower and more labile adsorption that is independent of gIII. After virions have been complexed with monoclonal antibodies against gIII (but not some monoclonal antibodies against other glycoproteins), both modes of adsorption were inhibited. Glycoprotein gIII affects virus stability and virus release, as well as adsorption. The effect on virus release is marked when the virus is defective in additional functions. Thus, although we found no obvious difference in the release of virus from gIII- or wild-type virus-infected rabbit kidney cells, release of a gIII-/gI- double mutant from the cells occurred less readily than did release of a gI- mutant. The gIII-/gI- and gIII- mutants, however, adsorbed to cells at a similar rate, indicating that the effects of gIII on adsorption and virus release constitute separate functions. The Bartha vaccine strain of pseudorabies virus has a defective gIII gene and is released poorly from rabbit kidney cells. After the resident Bartha gIII gene was replaced by the gIII gene of wild-type virus, virus release was enhanced considerably. Since inactivation of gIII in wild-type pseudorabies virus did not significantly affect virus release, the Bartha strain must be defective in another function which, in conjunction with gIII, significantly affects virus release. These results indicate again that gIII affects virus release in conjunction with other functions. Also, although the Bartha strain was functionally defective in virus release, it adsorbed to cells as well as wild-type virus did, showing that the effects of gIII on virus adsorption and release constitute separate functions. We conclude that gIII is a multifunctional glycoprotein.     

Early interactions of pseudorabies virus with host cells: functions of glycoprotein gIII.

Adsorption of mutants of pseudorabies virus (PrV) lacking glycoprotein gIII is slower and less efficient than is that of wild-type virus (C. Schreurs, T. C. Mettenleiter, F. Zuckermann, N. Snugg, and T. Ben-Porat, J. Virol. 62:2251-2257, 1988). To ascertain the functions of gIII in the early interactions of PrV with its host cells, we compared the effect on wild-type virus and gIII- mutants of antibodies specific for various PrV proteins. Although adsorption of wild-type virus was inhibited by polyvalent antisera against PrV as well as by sera against gIII and gp50 (but not sera against gII), adsorption of the gIII- mutants was not inhibited by any of these antisera. These results suggest that, in contrast to adsorption of wild-type PrV, the initial interactions of the gIII- mutants with their host cells are not mediated by specific viral proteins. Furthermore, competition experiments showed that wild-type Prv and the gIII- mutants do not compete for attachment to the same cellular components. These findings show that the initial attachment of PrV to its host cells can occur by a least two different modes--one mediated by glycoprotein gIII and the other unspecific. gIII- mutants not only did not adsorb as readily to cells as did wild-type virus but also did not penetrate cells as rapidly as did wild-type virus after having adsorbed. Antibodies against gIII did not inhibit the penetration of adsorbed virus (wild type or gIII-), whereas antibodies against gII and gp50 did. It is unlikely, therefore, that gIII functions directly in virus penetration. Our results support the premises that efficient adsorption of PrV to host cell components is mediated either directly or indirectly by gIII (or a complex of viral proteins for which the presence of gIII is functionally essential) and that this pathway of adsorption promotes the interactions of other viral membrane proteins with the appropriate cellular proteins, leading to the rapid penetration of the virus into the cells. The slower penetration of the gIII- mutants than of wild-type PrV appears to be related to the slower and less efficient alternative mode of adsorption of PrV that occurs in the absence of glycoprotein gIII.     

The putative cytoplasmic domain of the pseudorabies virus envelope protein gIII, the herpes simplex virus type 1 glycoprotein C homolog, is not required for normal export and localization.

Glycoprotein gIII of pseudorabies virus is a member of a conserved gene family found in at least seven diverse herpesviruses. We report here that the putative cytoplasmic domain of gIII is not required for transport to the cell surface and, unlike the prototype domain from herpes simplex virus type 1 glycoprotein C, is not required for stable membrane anchoring. Furthermore, this domain does not appear to be essential for incorporation of the glycoprotein into virions.     

Role of glycoprotein gIII of pseudorabies virus in virulence.

Deletion mutants of pseudorabies virus unable to express glycoprotein gIII, gI, or gp63 or double and triple mutants defective in these glycoproteins were constructed, and their virulence for day-old chickens inoculated intracerebrally was determined. Mutants of wild-type pseudorabies virus defective in glycoprotein gIII, gI, or gp63 were only slightly less virulent (at most, fivefold) for chickens than was the wild-type virus. However, mutants defective in both gIII and gI or gIII and gp63 were avirulent for chickens, despite their ability to grow in cell culture in vitro to about the same extent as mutants defective in gIII alone (which were virulent). These results show that gIII plays a role in virulence and does so in conjunction with gI or gp63. The effect of gIII on virulence was also shown when the resident gIII gene of variants of the Bartha vaccine strain (which codes for gIIIB) was replaced with a gIII gene derived from a virulent wild-type strain (which codes for gIIIKa); gIIIKa significantly enhanced the virulence of a variant of the Bartha strain to which partial virulence had been previously restored by marker rescue. Our results show that viral functions that play a role in the virulence of the virus (as measured by intracerebral inoculation of chickens) may act synergistically to affect the expression of virulence and that the ability of the virus to grow in cell culture is not necessarily correlated with virulence.     

Pseudorabies virus gIII and bovine herpesvirus 1 gIII share complementary functions.

The gIII glycoproteins of bovine herpesvirus 1 (BHV-1) and of pseudorabies virus (PRV) are structurally homologous. Both proteins also play preeminent roles in mediating virus attachment to permissive cells. To directly compare the functional relation between these glycoproteins, we constructed a recombinant BHV-1 in which the BHV-1 gIII coding sequence was replaced by the PRV gene homolog. The resultant recombinant virus efficiently expressed PRV gIII and then incorporated it into its envelope. The levels of PRV gIII expression and incorporation were equivalent to those achieved by the wild-type virus for BHV-1 gIII. The recombinant virus was fully susceptible to neutralization by anti-PRV gIII neutralizing antibody. In addition, the virus attachment and penetration functions, as well as the virus replication efficiency, which were lost by deleting the BHV-1 gIII gene, were restored by expressing the PRV gIII homolog in its place. These results demonstrated that PRV gIII and BHV-1 gIII share complementary functions.     

The gIII glycoprotein of pseudorabies virus is involved in two distinct steps of virus attachment.

The entry of herpesviruses into cells involves two distinct stages: attachment or adsorption to the cell surface followed by internalization. The virus envelope glycoproteins have been implicated in both stages. Pseudorabies virus attaches to cells by an early interaction that involves the viral glycoprotein gIII and a cellular heparinlike substance. We examined the role of gIII in the attachment process by analysis of a set of viruses carrying defined gIII mutations. The initial attachment of gIII mutants with an internal deletion of 134 amino acids (PrV2) to MDBK cells was indistinguishable from that of wild-type virus. The adsorption of these mutants was, however, much more sensitive than that of wild-type virus to competing heparin. Furthermore, while attachment of wild-type virus to MDBK cells led to a rapid loss of sensitivity to heparin, this was not the case with PrV2, which could be displaced from the cell surface by heparin after it had attached to the cells. We conclude that glycoprotein gIII is involved in two distinct steps of virus attachment and that the second of these steps but not the first is defective in PrV2.     

Release of pseudorabies virus from infected cells is controlled by several viral functions and is modulated by cellular components.

The role of the nonessential glycoproteins gI, gp63, and gIII in the release of pseudorabies virus from different cell lines was investigated. We show that these glycoproteins may have a beneficial or deleterious effect on virus release depending on the type of cell in which the virus is grown. Inactivation of the genes encoding either gI, gp63, or gIII has no detectable effect on virus release from rabbit kidney cells. Inactivation of gI or gp63 strongly promotes virus release from chicken embryo fibroblasts, whereas inactivation of gIII reduces virus release from these cells. A defect in both gI and gIII or in both gp63 and gIII diminishes virus release from rabbit kidney cells but improves release from chicken embryo fibroblasts. We demonstrate that all three nonessential glycoproteins contribute to one specific aspect of viral growth, namely, virus release, and that they affect virus release in conjunction with each other. Furthermore, our results show that the manifestation of the role of each of these viral functions in virus growth may differ in different cell types, i.e., that release is affected by these viral functions in conjunction with some unknown cellular function.     

Spontaneous fusions to prv43 can suppress the export defect of pseudorabies virus gIII signal peptide mutants.

We have devised an enrichment scheme for the isolation of export-competent derivatives of pseudorabies virus glycoprotein gIII signal peptide mutants. Enrichment is based upon a growth advantage imparted upon gIII-containing virions compared with virions lacking the glycoprotein. Each of identified derivatives suppressed the gIII signal peptide defect by fusing the gIII gene in frame to the prv43 gene that lay immediately upstream; the result was the synthesis of a Prv43-gIII hybrid protein. The deduced Prv43 protein is predicted to span a membrane multiple times, and it appeared that the gIII portion of each hybrid used a hydrophobic domain of Prv43 protein to initiate its export. For at least two of the isolates, the hybrid protein was efficiently translocated across the endoplasmic reticulum membrane but appeared to be poorly exported out of the endoplasmic reticulum. Nonetheless, the prv43-gIII fusions encoded a gIII species that was localized to the virus envelope. Because the gIII portion of each hybrid protein must be exposed on the virion surface to provide a growth advantage, our results also suggest a preliminary membrane topology for wild-type Prv43 protein.     

An amino-terminal deletion mutation of pseudorabies virus glycoprotein gIII affects protein localization and RNA accumulation.

We have constructed a pseudorabies virus mutant that contains virtually a complete deletion of the predicted signal sequence coding region for a nonessential envelope glycoprotein, gIII. No signal sequence mutants have been reported previously for a herpesvirus glycoprotein. Through endoglycosidase treatments and pulse-chase analysis, we have determined that the mutant gIII protein is not posttranslationally modified like the wild-type polypeptide, but rather is present as a single, stable species within the infected cell. The mutant polypeptide cannot be detected in the virus envelope, nor is it aberrantly localized to the tissue culture medium. Immunofluorescence studies have indicated that the mutant protein also is not localized to the surfaces of infected cells. In addition, Northern (RNA) and slot blot analyses, as well as in vitro translation experiments using infected-cell cytoplasmic RNA, have indicated that the mutant gIII allele is expressed at lower levels than the wild-type gene is. This is despite the fact that no alterations have been made upstream of the gIII coding sequence. From these results, it appears that the first 22 amino acids of the wild-type gIII protein define a necessary signal peptide that is responsible for at least the correct initiation of translocation and subsequent glycosylation of the gIII envelope glycoprotein within infected cells.     

The amino-terminal one-third of pseudorabies virus glycoprotein gIII contains a functional attachment domain, but this domain is not required for the efficient penetration of Vero cells.

We have examined the attachment and penetration phenotypes of several glycoprotein gIII mutants of pseudorabies virus (PRV) and have identified the first one-third of gIII as a region that mediates efficient virus attachment to PK15 and Vero cells. This portion of gIII, amino acids 25 through 157 of the wild-type sequence, appeared to support attachment by binding to heparinlike molecules on cell surfaces. Virions containing the first one-third of gIII were sensitive to heparin competition and showed greatly reduced infectivity on cells treated with heparinase. PRV virions lacking the first one-third of the mature glycoprotein exhibited only residual binding to cells if challenged by vigorous washing with phosphate-buffered saline at 2 h postinfection at 4 degrees C. This residual binding was resistant to heparin competition, and strains lacking the first one-third of gIII were able to infect cells treated with heparinase as effectively as untreated cells. When we determined the penetration phenotypes for each strain, we found that gIII-mediated virus attachment was necessary for timely penetration of PK15 cells but remarkably was not required for efficient virus penetration of Vero cells. Moreover, wild-type PRV was actually prohibited from rapid penetration of Vero cells by a gIII-heparan sulfate interaction. Our results indicate that initial virus binding to heparan sulfate via glycoprotein gIII is not required for efficient PRV infection of all cell types and may in fact be detrimental in some instances.     

Interaction of glycoprotein gIII with a cellular heparinlike substance mediates adsorption of pseudorabies virus.

Glycoprotein gIII is one of the major envelope glycoproteins of pseudorabies virus (PrV) (Suid herpesvirus 1). Although it is dispensable for viral growth, it has been shown to play a prominent role in the attachment of the virus to target cells, since gIII- deletion mutants are severely impaired in adsorption (C. Schreurs, T. C. Mettenleiter, F. Zuckermann, N. Sugg, and T. Ben-Porat, J. Virol. 62:2251-2257, 1988). We show here that during the process of adsorption of PrV, the viral glycoprotein gIII interacts with a cellular heparinlike receptor. This conclusion is based on the following findings. (i) Heparin inhibits plaque formation of PrV by preventing the adsorption of wild-type virions to target cells. However, heparin does not interfere with the plaque formation of PrV mutants that lack glycoprotein gIII. (ii) Wild-type virions readily adsorb to matrix-bound heparin, whereas gIII- mutants do not. (iii) Pretreatment of cells with heparinase reduces considerably the ability of wild-type PrV to adsorb to these cells and to form plaques but does not negatively affect gIII- mutants. (iv) Glycoprotein gIII binds to heparin and appears to do so in conjunction with glycoprotein gII. Although heparin significantly reduces the adsorption of wild-type virus to all cell types tested, quantitative differences in the degree of inhibition of virus adsorption by heparin to different cell types were observed. Different cell types also retain their abilities to adsorb wild-type PrV to a different extent after treatment with heparinase and differ somewhat in their relative abilities to adsorb gIII- mutants. Our results show that while the primary pathway of adsorption of wild-type PrV to cells occurs via the interaction of viral glycoprotein gIII with a cellular heparinlike receptor, an alternative mode of adsorption, which is not dependent on either component, exists. Furthermore, the relative abilities of different cell types to adsorb PrV by the gIII-dependent or the alternative mode vary to some extent.     

Mutations in the C-terminal hydrophobic domain of pseudorabies virus gIII affect both membrane anchoring and protein export.

The transmembrane and anchor region of pseudorabies virus gIII is postulated to be in the 35 hydrophobic amino acids (residues 436 to 470) found near the carboxy terminus of the 479-amino-acid envelope protein. In this study, we used a genetic approach to localize the functional gIII membrane anchor between amino acids 443 and 466. Mutant gIII proteins lacking the membrane anchor were not associated with virus particles, indicating that membrane retention is a prerequisite for virion localization. Unexpectedly, the specific hydrophobic gIII sequence defined by these deletions was not required for membrane anchor function since the entire region could be replaced with leucine residues without affecting gIII membrane retention, export, or virion localization. The hydrophobic region appears to encode more than the membrane anchor domain since both efficiency of posttranslational processing and localization to virions are affected by mutations in this region. We speculate that the composition of the hydrophobic domain influences the overall conformation of gIII, which in turn effects the efficiency of gIII export and processing. The virion localization phenotype is probably indirect and reflects the efficiency of protein processing. This conclusion provides insight into the mechanism of glycoprotein incorporation into virions.     

Characterization and mapping of a nonessential pseudorabies virus glycoprotein.

Antigenic variants of pseudorabies virus (PRV) containing mutations in a viral glycoprotein with a molecular weight of 82,000 (gIII) were isolated by selecting for resistance to a complement-dependent neutralizing monoclonal antibody (MCA82-2) directed against gIII. These mutants were completely resistant to neutralization with MCA82-2 in the presence of complement. Two mutants selected for further studies either did not express gIII or expressed an improperly processed form of the glycoprotein. The mutations were also associated with an altered plaque morphology (syncytium formation). The gIII gene was mapped by marker rescue of a gIII- mutant with cloned restriction enzyme fragments to the long unique region of the PRV genome between 0.376 and 0.383 map units. This corresponds to the map location of a glycoprotein described by Robbins et al. (J. Mol. Appl. Gen. 2:485-496, 1984). Since gIII is nonessential for viral replication in cell culture and has several other characteristics in common with the herpes simplex virus glycoprotein gC, gIII may represent the PRV equivalent to herpes simplex virus gC.     

Glycoprotein gI of pseudorabies virus promotes cell fusion and virus spread via direct cell-to-cell transmission.

Mutants of pseudorabies virus defective in either glycoprotein gI or gIII are only slightly less virulent for mice and chickens than is wild-type virus, while mutants defective in both gI and gIII are avirulent. To clarify the reason for the lack of virulence of the gI- gIII- mutants, we have analyzed in some detail the interactions of these mutants with their hosts. The results obtained showed that the gI glycoprotein is an accessory protein that promotes cell fusion. This conclusion is based on the findings that in some cell types, syncytium formation is significantly reduced in mutants deficient in gI. Furthermore, despite efficient replication, gI- mutants form significantly smaller plaques on some cell types. Finally, while wild-type and gI- virus are neutralized similarly by antisera, the size of the plaques formed by gI- mutants, but not by wild-type virus, is reduced by the presence of neutralizing antibodies in the overlay. Passive immunization of mice with neutralizing antipseudorabies virus sera is also considerably more effective in protecting them against challenge with gI- mutants than in protecting them against challenge with wild-type virus. These results show that gI- mutants are deficient in their ability to form syncytia and to spread directly by cell-to-cell transmission and that these mutants spread mainly by adsorption of released virus to uninfected cells. Wild-type virus and gIII- mutants, however, spread mainly via direct cell-to-cell transmission both in vivo and in vitro. We postulate that the lack of virulence of the gIII- gI- virus is attributable to its inability to spread by either mode, the defect in gIII affecting virus spread by adsorption of released virus and the defect in gI affecting cell-to-cell spread. Although a gI- gIII- mutant replicates as well as a gIII- mutant, it will be amplified much less well. Our results with in vitro systems show that this is indeed the case.     

Analysis of pseudorabies virus glycoprotein gIII localization and modification by using novel infectious viral mutants carrying unique EcoRI sites.

We have constructed two pseudorabies virus (PRV) mutants, each with a unique EcoRI restriction site in the nonessential gIII envelope glycoprotein gene. Since no natural PRV isolate has been reported to contain EcoRI sites, the isolation and single-step growth curve analysis of these mutants established that PRV can carry such a site with little ill effect in tissue culture. Virus carrying these defined mutations produced novel gIII proteins that enabled us to begin functional assignment of protein localization information within the gIII gene. Specifically, one viral mutant contained an in-frame synthetic EcoRI linker sequence that was flanked on one side by the first one-third of the gIII gene and on the other side by the last one-third of the gene. The resulting protein lacked the middle one-third of the parental species, including five of eight putative N-linked glycosylation signals, but was still glycosylated and found in enveloped virions; it was not secreted into the medium. A second viral mutant contained an in-frame synthetic EcoRI linker sequence that additionally specified a nonsense codon at position 158, producing a gIII protein that was glycosylated and secreted into the medium; the fragment was not found in enveloped virions. By endoglycosidase and pulse-chase analyses, we established a precursor-product relationship between the various forms of gIII expressed in the parental and mutant strains, and perhaps determined certain features of the gIII protein that are required for its efficient export within the cell.     

Expression of glycoprotein gIII-human decay-accelerating factor chimera on the bovine herpesvirus 1 virion via a glycosyl phosphatidylinositol-based membrane anchor.

Mutants of bovine herpesvirus 1 that express a truncated envelope glycoprotein gIII or a gIII-human decay-accelerating factor (hDAF) chimeric protein (gIII.hDAF) were employed to evaluate the function of the transmembrane and cytoplasmic domains of the gIII molecule. Truncated gIII (i.e., lacking the transmembrane and cytoplasmic region) was readily released from infected cells and was not detected on mature virus particles. In contrast, replacement of the transmembrane and cytoplasmic domains with the carboxyl-terminal portion of hDAF restored the expression of gIII on the membranes of infected cells as well as on virion surfaces. The presence of the gIII.hDAF chimera on virus particles was also associated with normal gIII function, i.e., the mediation of virus attachment and penetration. The gIII-hDAF chimera, which is present on both infected cell surfaces and virions, could be cleaved by a phosphatidylinositol-specific phospholipase C, indicating that it was anchored in the membrane via glycosyl phosphatidylinositol. Our results from this study suggest that the transmembrane and cytoplasmic regions of the gIII molecule serve as a general membrane anchor, but they do not contain structural signals required for the specific assembly of envelope proteins into mature virions.     

Synthesis, cellular location, and immunogenicity of bovine herpesvirus 1 glycoproteins gI and gIII expressed by recombinant vaccinia virus.

Two of the major glycoproteins of bovine herpesvirus 1 (BHV-1) are gI, a polypeptide complex with apparent molecular weights of 130,000, 74,000, and 55,000, and gIII (a 91,000-molecular-weight [91K] glycoprotein), which also exists as a 180K dimer. Vaccinia virus (VAC) recombinants were constructed which carry full-length gI (VAC-I) or gIII (VAC-III) genes. The genes for gI and gIII were each placed under the control of the early VAC 7.5K gene promoter and inserted within the VAC gene for thymidine kinase. The recombinant viruses VAC-I and VAC-III retained infectivity and expressed both precursor and mature forms of glycoproteins gI and gIII. The polypeptide backbones, partially glycosylated precursors, and mature gI and gIII glycoproteins were indistinguishable from those produced in BHV-1-infected cells. Consequently, they were apparently cleaved, glycosylated, and transported in a manner similar to that seen during authentic BHV-1 infection, although the processing efficiencies of both gI and gIII were generally higher in recombinant-infected cells than in BHV-1-infected cells. Immunofluorescence studies further demonstrated that the mature gI and gIII glycoproteins were transported to and expressed on the surface of cells infected with the respective recombinants. Immunization of cattle with recombinant viruses VAC-I and VAC-III resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gI and gIII. These data demonstrate the immunogenicity of VAC-expressed gI and gIII and indicate the potential of these recombinant glycoproteins as a vaccine against BHV-1.